Ion channels are exquisite molecular machines. By opening and closing an ion selective pore across the cell membrane, these proteins ultimately control everything from our senses to our thoughts. Our long term goal is to understand the precise molecular motions that underlie this gating behavior of ion channels. Cyclic nucleotide-gated (CNG) channels produce the primary electrical signal in our photoreceptors in response to light. They are nonselective cation channels that are opened by the direct binding of cyclic nucleotides (cAMP and cGMP) to the channel and modulated by various second messengers. In addition to their role in vision, they are also essential for olfaction and taste, and mutations in these channels cause an assortment of sensory disorders ranging from blindness to anosmia. Their dynamic behavior controls our visual perception, yet the molecular mechanism for their function is largely unknown. This void is due, in part, to a lack of experimental approaches that allow us to watch proteins in action in real time at atomic resolution. We aim to fill this void by developing novel fluorescence approaches and applying them to investigate the mechanisms of activation and modulation of CNG channels. We will take advantage of two exciting new developments: 1) our solution of the x-ray crystal structures of the intracellular ligand binding and gating domains of the closely related HCN2 and SpIH channels, and 2) our development of methods for simultaneous current and fluorescence measurements from cell-free membrane patches (termed patch-clamp fluorometry, PCF). Our specific aims are to precisely determine the molecular rearrangement in two important parts of the channel, the cyclic nucleotide- binding domain, and the C-linker, the region that couples binding of cyclic nucleotides to opening of the pore. At the conclusion of these experiments we will know a great deal more about how CNG and related channels work, and will have fully developed new approaches to studying molecular rearrangements applicable to other channels and other proteins. PUBLIC HEALTH RELEVANCE: Ion channels are the transistors of the brain and thereby control everything from our senses to our thoughts. Cyclic nucleotide-gated (CNG) channels produce the primary electrical signal in our rods and cones in response to light, and mutations in these channels cause an assortment of sensory disorders ranging from blindness to anosmia. Our long term goal is to understand how these important proteins work at the molecular level to uncover basic mechanisms of protein function and enable us to develop targeted therapies for neurological diseases.